Cross-regulated assembly of a two-component copolymer determines bacterial cell curvature

Abstract

Many bacterial proteins perform functions that require them to properly assemble into higher order structures. Often this assembly process depends on energy sources, as is the case for most cytoskeletal elements in the cytoplasm. It was recently discovered that cytoskeleton-like filaments can also assemble in the periplasm of Gram-negative bacteria, which lacks ATP or GTP. In the comma shaped, gram negative bacterial species, Vibrio cholerae, two proteins called CrvA and CrvB promote cell curvature by assembling into a long filamentous structure in the periplasm. CrvA and CrvB participate in regulating one another’s levels and assembly to form a periplasmic copolymer. This CrvAB structure localizes along the shorter curved face of the bacteria and upon heterologous expression is sufficient to curve all Gram-negative bacterial species tested. Thus, CrvAB appears to form an autonomous, periskeletal module sufficient for curvature induction. This thesis will describe our multidisciplinary approach toward a better understanding of how CrvA and CrvB interact and regulate one another. CrvB is homologous to CrvA at its N-terminus but has an additional large CrvB-specific domain at its C-terminus. Cells lacking either crvA or crvB exhibit a straight-rod morphology and differ in the molecular assembly of the remaining gene product. While CrvA is capable of assembling to some degree in cells lacking crvB, CrvB assemblies are absent upon deletion of crvA. To understand the molecular and structural basis of these interactions CrvA and CrvB have been purified and their assembly, alone and in combination, was examined in vitro. Preliminary biochemical and electron microscopy studies suggest a new copolymer assembly model. Here I will describe these findings and their implications for the general processes of energy-independent filament assembly and its role in bacterial cell shape determinant.